The long term objective of this proposal is the elucidation of nucleotide excision repair (NER) mechanisms in eucaryotic systems. NER is one of a small number of biochemical pathways that maintain the chemical integrity of the genetic material by a constant monitoring and repair process. Defects in NER have been found to be responsible for the development of several human diseases including xeroderma pigmentosum (XP), Cockayne's syndrome, and trichothiodystrophy. In addition, defects in NER accelerate carcinogenesis and may be involved in the aging process. The NER pathway is complex in eucaryotes involving perhaps as many as thirty different genes for the complete repair process. The focus of this project is primarily on the early stages of NER which involve the recognition of damage, priming of the damaged site for subsequent incision and the incision step itself. These early steps appear to involve about twelve to fifteen genes. A significant number of these genes have been cloned and these accomplishments have set the stage for more detailed biochemical studies. The specific aims of the proposed project are to: l) investigate the function of the XPC gene in NER 2) further investigate and determine the functional relevance of previously demonstrated protein-protein interactions among DNA repair factors, and 3) analyze recently isolated novel genes to determine if they have an involvement in either NER or recombinational repair pathways. These studies will be conducted using a combination of biochemical and genetic approaches including the two-hybrid system, cell free repair assays, in vitro assays to detect protein-protein interactions, and site directed mutagenesis.